This revised R21 is in response to the PA entitled "Directed Stem Cell Differentiation for Cell-Based Therapies for Heart, Lung, and Blood, and Aging Diseases". This grant is based on the premise that endothelial dysfunction underlies the pathophysiology of vascular impairment associated with conditions (hypertension, hyperlipidemia, obesity, aging) and diseases (diabetes, cardiovascular disease). Vascular repair is partially mediated by the recruitment of circulating endothelial progenitor stem cells (EPCs) and the central theme of this proposal is that EPCs represent circulating post-fetal (adult) vasopotent stem cells that are involved in maintaining vascular health and preventing disease. Our long-term goal is to develop EPC-based therapies for vascular disease and our specific aims will provide fundamental information regarding the biology of these cells. This knowledge is an absolute prerequisite for the rational development of cell- based therapies using vascular progenitor cells, an approach that holds tremendous potential for treating a host of life-threatening diseases. The main hypothesis to be tested is that primordial EPCs and mature endothelial cells are inherently biologically different and each requires a unique environmental milieu to control proliferation and differentiation. Our studies will exploit the availability of GFP transgenic swine as a source of cells. Aim 1 is to isolate EPCs from the peripheral circulation of adult animals employing novel cell culture methods to establish clonal lines of proliferating, but undifferentiated, cells. Aim 2 is to establish culture conditions for proliferation using unique methods, including suspension culture and selected growth factors and cytokines, to provide the environment for robust growth while suppressing differentiation. Aim 3 is to characterize EPCs in terms of function and molecular markers associated with differentiation state and to define the conditions that induce EPC differentiation into mature endothelial cells using combinations of extracellular matrices and growth factors/cytokines. This project will provide crucial insight into the mechanisms that control differentiation and will yield novel adult vascular progenitor cell lines for the eventual development of cell-based angiogenesis or vasculogenesis for reversing vascular disease. This work is highly relevant to human health. Heart and blood vessel disorders are major killers of people of all genders and ages and the work described here will lead to new treatments for these diseases. [unreadable] [unreadable] [unreadable]